HLA A*24:02–restricted T cell receptors cross-recognize bacterial and preproinsulin peptides in type 1 diabetes

CD8+ T cells destroy insulin-producing pancreatic β cells in type 1 diabetes through HLA class I–restricted presentation of self-antigens. Combinatorial peptide library screening was used to produce a preferred peptide recognition landscape for a patient-derived T cell receptor (TCR) that recognized the preproinsulin-derived (PPI-derived) peptide sequence LWMRLLPLL in the context of disease risk allele HLA A*24:02. Data were used to generate a strong superagonist peptide, enabling production of an autoimmune HLA A*24:02–peptide–TCR structure by crystal seeding. TCR binding to the PPI epitope was strongly focused on peptide residues Arg4 and Leu5, with more flexibility at other positions, allowing the TCR to strongly engage many peptides derived from pathogenic bacteria. We confirmed an epitope from Klebsiella that was recognized by PPI-reactive T cells from 3 of 3 HLA A*24:02+ patients. Remarkably, the same epitope selected T cells from 7 of 8 HLA A*24+ healthy donors that cross-reacted with PPI, leading to recognition and killing of HLA A*24:02+ cells expressing PPI. These data provide a mechanism by which molecular mimicry between pathogen and self-antigens could have resulted in the breaking of self-tolerance to initiate disease.


HLA A*24 typing of donors with antibody
For the HLA typing of healthy donors an unconjugated anti-HLA A*24 antibody (catalog number H6098-29, clone 4i94, United States Biological, Salem, MA, USA) was used with a goat anti-mouse PE conjugated secondary antibody (catalog number 550589, BD Biosciences).The primary antibody also binds to HLA A*23, so the 1/8 donors that we failed to generate HLA A*24 restricted T-cells from may have been HLA A*23 + and not HLA A*24 + .

Antigen presenting cells
The following cell lines were sourced locally with ATCC references included to act as a resource for culture and validation guidelines: the B-lymphoblastoid C1R (ATCC® CRL-1993™), acute monocytic leukaemia THP-1 (ATCC® TIB-202™) and lung carcinoma A549 (ATCC® CCL-185™), with each being cultured in R10 media.C1R and THP-1 cells were grown in suspension and split 1:10 once or twice a week.A549 are adherent and passaged by detachment with D-PBS and 2 mM EDTA and split 1:10-1:20 once a week.C1R, THP-1 and A549 cells are naturally HLA A*24:02 negative and therefore transduced with HLA A*24:02 to act as antigen presenting cells for the study.The fes and gsiA genes from Klebsiella oxytoca strains, encoding Enterochelin esterase containing the RYPRLFGIV sequence and gsiA encoding Glutathione ABC transporter ATP-binding protein containing the SLPRLFPLL sequence respectively (Supplementary Figure 8A) were lentivirally transduced for epitope validation experiments (see below).Briefly, codon optimised HLA A*24:02, fes and gsiA genes were synthesised (Geneart, Life Technologies, Carlsbad, California, US) and cloned in-house to a third-generation lentiviral plasmid (p), SnapFast (pSF) (Oxgene, Oxford Genetics Ltd, Littlemore, Oxford, UK).The fes and gsiA genes were expressed with a rat (r) CD2 co-marker: XbaI-Kozakgene-XhoI-P2A-rCD2-SalI-Stop, whereas HLA A*24:02 did not require the rCD2 co-marker as it is expressed at the cell surface: XbaI-Kozak-gene-Stop-SalI-Stop.C1R cells were also transduced with the IGF2BP2 gene encoding IMP2 as previously described (45) with a rCD2 co-marker, to act as an irrelevant protein for the Klebsiella epitope validation experiments.For transfection, pSF (1.52 μg), envelope plasmid (pMD2.G; 0.72 μg) and packaging plasmids (pMDLg/pRRE; 1.83μg and pRSV-REV; 1.83μg) were mixed in 300 μL of Opti-MEM, reduced serum medium (Thermo Fisher Scientific) followed by mixing with 1 μg/mL Polyethylenimine (PEI; Merck) at a 3:1 PEI: plasmid ratio.Plasmid/PEI mixtures were incubated at room temperature for 15 min, added dropwise to locally sourced HEK293T cells (80% confluence in one well of a 6-well plate) and incubated at 37°C in a 5% CO2 humidified atmosphere.The supernatants containing lentiviral vectors were harvested, passed through a 0.4 μm filter and used immediately for transduction.Cells (0.1-0.2 x10 6 per well of a 24 well plate) were transduced by spinfection with 1 mL of viral supernatant and 1 mL of media at 800 x g for 1.5 h at room temperature with 0.5 mg/mL of LentiBOOST (Sirion Biotech, Cambridge, MA, USA), then incubated at 37°C overnight and media replaced the following morning.For C1R cells and transduction of HLA A*24:02, expression was confirmed one week post transduction by staining with anti HLA A*24 PE conjugated antibody (catalog number LS-C179732-100, clone 17A10, LsBIO, Seattle, WA, US) and a clone procured by limiting dilution in 96U well plates.Expression of HLA A*24 for THP-1 and A549 cells was confirmed using anti-HLA A*24 antibody clone 4i94 as above, and cells sorted via a BD FACS Aria III (Central Biotechnology Services, Cardiff University) to achieve cell lines with >95% HLA A*24 expression.Cell lines expressing gsiA, fes or IGF2BP2 were enriched via the rCD2 co-marker using 0.02 mg of a mouse anti-rCD2 PE conjugated antibody (catalog number 201305, clone OX-34, BioLegend) per 0.1-10 x10 6 cells in 50-100 μL of MACS buffer (D-PBS, 0.5% BSA and 2 mM EDTA), followed by magnetic enrichment using anti-PE microbeads according to the manufacturer's instruction (Miltenyi Biotec).K-562 cells (ATCC® CCL-243™), expressing HLA A*A24 and preproinsulin were generated before and cultured in R10 as suspension cells with selection antibiotics (14).All cell lines were tested for mycoplasma, as above.

Sizing scan and PS-CPL screening
The peptide length-preference of the 4C6 T cell clone was determined by 'sizing scan' as previously described (21).4C6 T-cells were rested overnight in R5 media (as for R10 but with 5% FBS) to reduce spontaneous activation, followed by incubation in the presence of C1R-HLA A*24:02, with peptide mixtures of defined length and random amino acid sequence (X 8-14 in which X is any of the 20 proteogenic L-amino acids, excluding cysteine) in R5 media overnight at 37°C and 5% CO2.MIP-1β production was measured by enzyme-linked immunosorbent assay (ELISA, R&D Systems, Bio-Techne, Minneapolis, MN, US) as a proxy for T cell activation as previously described (22).Peptide mixtures were synthesised at >40% purity (Pepscan Presto, Lelystad, Netherlands) and stored at -80°C in DMSO at 40 mM.Mixtures were used in assays at a concentration of 1 mM.Following conformation of the preference of 4C6 T-cells for 9-mer peptides, nonamer PS-CPL screening was performed using libraries synthesised at >40% purity as described previously (22,23,43).Libraries were stored at -80°C in DMSO at 20 mM.For assays, 6×10 4 C1R-HLA A*24:02 were pulsed with 100 μM of each CPL library mixture for 2 h at 37°C.Following this, 3×10 4 4C6 T cells were co-incubated with the pulsed C1R-HLA A*24:02 cells overnight at 37°C and 5% CO2.
Assay conditions were performed in duplicate.Supernatants were harvested the following morning and assayed for the presence of MIP-1β, as above, by ELISA.

Positional scanning combinatorial libraries (PS-CPL) for identification of agonist peptides
CPL-based importance sampling, as described in (20), was used to sample peptides from the entire peptide universe with bias toward good agonists.Additionally, PS-CPL screening data from 4C6 T-cells was used to probe a human pathogen proteome database to produce a ranked list of likely peptide ligands, using a Top-ranking candidate peptides were ordered in crude form (>40% purity; GLS Biochem, Shanghai, China).
Confirmed peptide agonists were subsequently ordered in pure form (>95% purity, Peptide Synthetics, Hampshire, UK).Reactivity of 4C6 T-cells to candidate peptides was assessed by ELISA as previously described (43).Assays conditions were performed in duplicate and EC50 values were generated using GraphPad Prism software analysis (v.9.0.1).

Production of soluble protein
Soluble TCR and pMHC proteins were manufactured as previously described (47).Briefly, codon-optimised synthetic genes for 4C6 TCR α chain, TCR β chain, HLA A*24:02 heavy chain (α1, α2, and α3, domains) and Beta-2-Microglobulin (2M) chain were generated by Geneart.Sequences were confirmed by Sanger DNA sequencing (Eurofins).TCR expression constructs were designed to contain a non-native disulphide bond, by substituting residues 48 and 57 of TCR α chain and TCR β chain constant domains respectively with cysteine residues, in order to produce stable soluble TCRs (28).The expressed protein sequence is shown in Supplementary Figure 1.For production of biotinylated pMHC monomers, a biotin tag (GLNDIFEAQKIEWHE) was added to the C-terminus of the HLA A*24:02 heavy chain construct.The TCR α chain, TCR β chain, the HLA A*24:02 heavy chain and 2M constructs were inserted into separate pGEM-T7 expression plasmids (Promega, Madison, WI, USA) under the control of the T7 promoter.Competent BL21 E. coli cells (Thermo Fisher Scientific) were used to produce proteins in the form of soluble inclusion bodies, using 0.5 mM Isopropyl β-d-1-thiogalactopyranoside (IPTG, Neo Biotech Nanterre, France) to induce expression.Inclusion bodies were chemically refolded to form TCR or pMHC complexes as described previously (47).

Surface plasmon resonance (SPR)
SPR equilibrium binding analysis was performed using a BIAcore T200 (Cytiva, Marlborough, MA, US) equipped with a CM5 sensor chip as previously reported (22, 33).H2-K d -SIINFEKL was used as a negative control on flow cell 1.SPR kinetic analyses were carried out to determine the KD values for the TCR, at 25°C.For all kinetic experiments, approximately 500 RUs of biotinylated pMHC was coupled to the CM5 sensor chip surface.Ten TCR samples, made in serial dilution from a starting concentration of 361.5 µM, were sequentially injected over the chip at 30 µL/min.KD values were calculated assuming 1:1 Langmuir binding [AB=B * ABMAX/(KD + B)] and the data were analysed using a global fit algorithm (GraphPad Prism).For thermodynamics experiments, the SPR method was repeated at 8, 15, 21, 25, 35, and 40°C.The binding free energies (ΔG = RT ln KD) were plotted against temperature and the thermodynamic parameters (ΔH° and TΔS°) were calculated using the non-linear Van't Hoff equation (RT ln KD = ΔH° -TΔS° + ΔCp°(T -T°) -TΔCp° ln (T/T°)) with T° = 298 K)

Klebsiella oxytoca epitope validation
The 4C6 T-cell clone was rested overnight in R5 media, as above then incubated with antigen presenting cells, with 3 x10 4 4C6 T-cells and 6 x10 4 antigen presenting cells per well of a 96U well plate.The cells were incubated overnight in R5 media at 37°C and 5% CO2, with all conditions performed in triplicate.

Peptide priming of CD8 T-cells
CD8 T-cells from an HLA A*24 + healthy donor (BB51) were magnetically enriched by positive selection using CD8 microbeads according to the manufacturer's instructions (Miltenyi Biotec).CD8 negative cells from the same donor were irradiated then combined with the CD8 T-cells in 24 well plates, with 8 x10 6 and 3 x10 6 cells per well respectively.Cells were cultured in 2 mL of T-cell media per well, recipe as above, but with 20 IU/mL of IL-2 and no IL-15.The media (50%) was changed thrice weekly.For the peptide primed conditions Klebsiella-SLPRLFPLL or PPI-LWMRLLPLL peptides were added directly to the wells at a final concentration of 10 -5 M. The unprimed well received an equivalent volume of DMSO to that of the peptide primed conditions. 2 μg/mL of Ultra-LEAF TM anti-CD28 antibody (catalog number 302934, clone CD28.2, BioLegend) was added at the same time as the peptides or DMSO.After 2 weeks the lines were stained with HLA A*24:02 irrelevant-AYAAAAAAL, Klebsiella-SLPRLFPLL and PPI-LWMRLLPLL tetramers.

Magnetic-based pMHC tetramer enrichment from HLA A*24 + patients and healthy donors
For type 1 diabetic patient CCPO-1406, 16.3 x10 6 freshly isolated PBMCs were treated with 50 nM PKI (26) for 30 min at 37°C then labelled with 0.5 µg PE conjugated PPI-LWMRLLPLL tetramer per 3 x10 6 cells for 30 min on ice.Excess tetramer was washed from the cells with ice-cold MACS buffer, then tetramer labelled cells enriched using anti-PE magnetic microbeads according to the manufacturer's instructions (Miltenyi Biotec) and as previously described (14).Positive cells from the column were incubated overnight in a well of a 96U plate then expanded the next day in situ with mixed irradiated (3100 cGy) PBMCs from three donors and 1 μg/mL of PHA.As the cells expanded, they were divided between 96U wells then transferred to wells of a 48 well plate and kept at a 0.5-1 x10 6 per well.The cells were re-stained with HLA A*A24 Klebsiella-SLPRLFPLL and PPI-LWMRLLPLL tetramers, using PKI pre-treatment and tetramer staining stabilisation with an anti-PE unconjugated antibody (catalog number 408102, clone PE001, BioLegend) (53).cell media with CD3/CD28 Dynabeads according to the manufacturer's instructions (ThermoFisher Scientific), followed by 42 x10 6 of the expanded T-cells undergoing enrichment with PE conjugated PPI-LWMRLLPLL tetramer, then expansion and staining with tetramers as described above.
For the healthy donors, cryopreserved PBMCs (30.1-90.6 x10 6 cells, mean 55.9 x10 6 ) were defrosted and used for tetramer enrichment.Whereas T-cells from the type 1 diabetic donors were enriched with PPI-LWMRLLPLL tetramer, the healthy donors were enriched with either Klebsiella oxytoca-SLPRLFPLL or HLA A*24:02 CMV-AYAQKIFKIL tetramers, using the same approach as described above for CCPO-1406.
The process of magnetic enrichment with tetramers followed by expansion was performed once (T1D-12, BB51, BB52, 572D) or twice (CCPO-1406, BB31, BB25, BB64, BB57), depending on the number of cells available and percentage of tetramer staining seen after the first enrichment.Central Biotechnology Services at Cardiff University and gated as follows: lymphocytes (FSC-A versus SSC-A), single cells (FSC-A versus FSC-H), viable (LIVE/DEAD™ fixable violet dead cell stain low/negative) CD3 + cells, CD8 + cells, then PE versus APC tetramer.Irrelevant AYAAAAAAL-PE versus AYAAAAAAL-APC tetramer staining was used to set the sorting gate.Tetramer + cells were collected directly into 300 μL of RNAprotect cell reagent (Qiagen, Hilden, Germany) according to the manufacturer's instructions, then used for TCR sequencing.RNA extraction was carried out using the RNeasy Micro kit (Qiagen).cDNA was synthesized using the 5′/3′ SMARTer kit according to the manufacturer's instructions (Takara Bio, Paris, France).The SMARTer approach used a Murine Moloney Leukaemia Virus (MMLV) reverse transcriptase, a 3′ oligo-dT primer and a 5′ oligonucleotide to generate cDNA templates flanked by a known, universal anchor sequence at the 5′.A Step-Out PCR was performed using a pair of primers consisting of 3' TRAC or TRBC-specific reverse primer (Eurofins Genomics) and a 5' universal anchor-specific forward primer (Takara Bio).All samples were used for the following PCR reaction: 2.5 μL template cDNA, 0. 5 μL High Fidelity Phusion Taq polymerase, 10 μL 5X Phusion buffer, 0.5 μL DMSO, 1 μL dNTP Mix (stock concentration of 10 mM of each) (all from Thermo Fisher Scientific), 1 μL of TRAC or TRBC-specific primer (10 μM stock), 5 μL of 10X anchor-specific universal primer (Takara Bio), and nuclease-free water for a final reaction volume of 50 μL (cycling conditions: 5 min at 94°C, 30 cycles of 30 s at 94°C, 30 s at 63°C for alpha chains or 30 s at 66°C for beta chains, 120 s at 72°C).Subsequently, 5 μL of the Step-out PCR products were taken for a nested PCR, using 1 μL of barcoded forward (universal) and reverse (TRAC or TRBC) primers (10 μM stock) (Eurofins Genomics), 0.5 μL High Fidelity Phusion Taq polymerase, 10 μL 5X Phusion buffer, 0.5 μL DMSO, 1 μL dNTP Mix (stock concentration of 10 mM each) and nuclease-free water for a final reaction volume of 50 μL (cycling conditions: 5 min at 94°C, 30 cycles of 30 s at 94°C, 30 s at 62°C, 120 s at 72°C, and a final 10 min at 72°C).The final PCR products were loaded on a 1% agarose gel and purified with the Monarch ® gel extraction kit (New England Biolabs, Ipswich, MA, US).Purified products were sequenced on an Illumina MiSeq instrument using the MiSeq v2 reagent kit (Illumina, Cambridge, UK) according to the manufacturer's instructions.Sequence analysis was performed using MiXCR software (v3.0.7) (54).TCRs appearing in both SLPRLFPLL and LWMRLLPLL tetramer sorted populations were deemed as crossreactive.

Flow cytometry-based T-cell activation assays
T-cells were rested in R5 (as for R10 with 5% FBS) for 24 h before setting up the assay to help reduce spontaneous activation.For TNF-processing inhibitor (TAPI)-0 (Santa Cruz Biotechnology TX, US) assays, T-cells (3 x10 4 ) and target (6 x10 4 ) cells were co-incubated for 4-6 h with 30 μM TAPI-0 (Merck), and with antibodies directed against TNF (catalog number 130-120-492, clone cA2, PE-Vio770 conjugated, Miltenyi Biotec) and CD107a (clone H4A3, FITC conjugated catalog number 555800, or PE conjugated catalog number 555801, BD Biosciences), with the latter detecting activation-induced degranulation of cytotoxic Tcells.Following co-incubation, cells were washed then stained with LIVE/DEAD™ fixable violet dead cell stain for 5 min at RT, FcR blocking reagent used according to the manufacturer's instructions (Miltenyi Biotec), and without washing antibodies against CD3 PerCP (as above) and CD8 APC (as above) added and incubated for 20 min on ice.Cells were washed in PBS and acquired immediately on a flow cytometer or fixed with 2% formaldehyde for acquisition within 24 h.Acquisition was performed on a ACEA NovoCyte 3005 with NovoSampler pro (ACEA, Agilent, Santa Clara, CA, USA).Gating was for lymphocytes (FSC-A/H versus SSC-A/H), single cells (FSC-A versus FSC-H), viable CD3 + cells (LIVE/DEAD™ fixable violet dead cell stain low/negative), CD3 + /CD8 + , then displayed as TNF versus CD107a.

Cytotoxicity assays
Chromium 51 ( 51 Cr) assays were performed using K-562 cells expressing HLA A*24:02 with and without preproinsulin as target cells for T-cell lines from donor BB57.K-562 cells were labelled with 51 Cr as sodium chromate (Perkin Elmer, Waltham, MA) and incubated at 37°C for 1 h then washed in R10 medium and incubated in 2 mL R10 medium for a further hour to allow excess 51 Cr to leach from the cells.Target cells (2,000 per well) were incubated with T-cell lines to give desired T-cell to K-562 cell ratios.Targets were incubated alone or with lysis buffer (5% Triton in H2O) to give spontaneous and maximum release of 51 Cr respectively.After incubation (6 h), culture supernatant was harvested and mixed with 150 μL of Opitphase Supermix scintillation cocktail (PerkinElmer) and the release of 51 Cr measured using a 1450 MicroBeta TriLux (PerkinElmer).Percentage killing was determined using the following calculation: publicly accessible webtool pioneered by Szomolay et al. (23) and refined by Whalley and colleagues (22).

%
killing = 100 − (( release from K-562 with T-cells − spontaneous release from K-562 total release from K-562 − spontaneous release from K-562 ) × 100) Supplementary Figure 1: Sequence of the 4C6 TCR.(A) Native nucleotide sequence of TCRa and TCRb genes showing VJ assignment (unequivocal assignment of TRBD gene not possible).(B) Bacterially expressed protein sequences manufactured in E.coli and refolded to make soluble 4C6 TCR for biophysical and structural studies.Sequences include non-native cysteine residues as indicated in bold underlined red text to form non-native disulphide bonding between the TCR a and b constant domains.Two other substitutions in the TCRb chain that aid refolding are indicated in bold underlined text.The Cys to Ala substitution was included to remove the possibility of incorrect disulphide bind formation.
cross-reacts with fungus derived peptides.Positional-scanning combinatorial peptide library data for 4C6 was used to screen a database of disease fungal species and the top 20 peptides selected for testing.(A) Peptide titrations using 4C6 and the top 20 fungal peptides (listed in B).Incubation overnight with C1Rs expressing HLA A*24:02 as antigen presenting cells.Assay supernatants used for MIP-1β ELISA.Data points shown for duplicate conditions.(B) Peptide sequence and origin.Scoring indicates prediction of how likely the peptide is to be recognized by 4C6 T-cells, with the best scoring peptide at the top.EC 50 of activation in bold indicate peptides seen more sensitively than the preproinsulin peptide in functional assays.

Supplementary Figure 7 :Supplementary Figure 8 :
Comparative sensitivity of 4C6 T-cells to preproinsulin, superagonist and Klebsiella oxytoca peptides.Sensitivity of 4C6 T-cells to preproinsulin (LWMRLLPLL), super-agonist (QLPRLFPLL) and Klebsiella oxytoca (SLPRLFPLL and RYPRLFGIV) peptides in a titration assay.Underlined amino acid residues are the same as the preproinsulin peptide.Residues in bold are present in the superagonist.Incubation overnight with C1R-HLA A*24:02 as antigen presenting cells.Assay supernatants used for MIP-1β ELISA.Data points shown for duplicate conditions.esterase (DUF3327 containing protein): fes gene Glutathione ABC transporter ATP-binding protein: gsiA gene 2RYPRLFGIV peptide (fes) 6. SLPRLFPLL peptide (gsiA) 1. WT 2. +HLA A*24:02 3. +HLA A*24:02 + fes 4. +HLA A*24:02 + gsiA Key: Preproinsulin and Klebsiella oxytoca crossreactive T-cells are present in multiple patients and the Klebsiella derived peptides are genuine epitopes.(A) Amino acid sequences of two Klebsiella oxytoca proteins with respective epitopes (RYPRLFGIV and SLPRLFPLL) highlighted in color and underlined.Genes (fes or gsiA) encoding these proteins were used for lentiviral transduction of antigen presenting cells for epitope validation.(B) Overnight activation assays with HLA A*24:02 restricted preproinsulin CD8 T-cell clone 4C6 with THP-1 or A549 cell lines co-transduced with HLA A*24:02 and fes or gsiA from Klebsiella oxytoca.Exogenous RYPRLFGIV and SLPRLFPLL peptides used at 10 -5 M. Supernatants used for MIP-1β (left) or TNF (right) ELISAs, with error bars depicting the standard deviation of triplicate conditions.MIP-1β or TNF production for T-cell alone conditions have been subtracted from the displayed data.Data using C1R antigen presenting cells shown in Figure 5. (C) T-cell lines created from HLA A*24 + type 1 diabetic patients by enrichment with HLA A*24:02 preproinsulin (PPI, LWMRLLPLL) tetramers were subsequently stained with Klebsiella (Kleb)-SLPRLFPLL or PPI tetramers.
: Crossreactive T-cell receptors recognizing Klebsiella and preproinsulin bear CDR1 and CDR3 similarities to the 4C6 TCR.T-cell lines from healthy HLA A*24 + donors, by enrichment detailed in (A), were subsequently sorted by flow cytometry with on Kleb or PPI tetramers and next generation sequencing performed for TCR clonotyping.TCRs shared between the Kleb and PPI sorted T-cells are shown, with their frequency from each tetramer sorted population shown on the left.The TCR sequence of the 4C6 clone is included for comparison, with residues that make contacts with the PPI peptide highlighted with colored circles.Alpha TCR chain similarities to the 4C6 TCR and between TCRs from the donors are colored according to the key.The specific allele for TRAJ37 is indicated, as *01 and *02 encode for a Glycine or Serine respectively at the same position (G-S-G/S-N-T-G-K-L-I-F).
Top ten scoring peptides from 500 'randomly' selected peptides based on the PS-CPL of 4C6, tested in comparison to the preproinsulin peptide LWMRLLPLL.Incubation overnight with C1Rs expressing HLA A24:02 as antigen presenting cells.Crude (>40%) purity peptides used for screening.Assay supernatants harvested for MIP-1β ELISA.Data points shown for duplicate conditions.EC 50 values in bold indicated peptides seen more sensitively than the preproinsulin peptide.NC = EC 50 not calculated.
Supplementary Figure 2: Identification of a superagonist peptide for 4C6 T-cells.